Immobilized microbial agent and application thereof in degradation of butyl xanthate and benzohydroxamic acid pollution

Abstract

The invention discloses an immobilized microbial inoculum and application thereof in degradation of butyl xanthate and benzohydroxamic acid pollution, a preparation method of the immobilized microbial inoculum is characterized by comprising the following steps: mixing biochar with a pseudomonas and cupriavidus composition, and fully adsorbing to obtain a product 1; fully and uniformly mixing a sodium alginate aqueous solution with the product 1 to obtain a product 2; dropwise adding the product 2 into a CaCl2 solution, fully curing, and purifying to obtain the product. According to the immobilized microbial agent disclosed by the invention, flora embedded with pseudomonas and Cupriavidus can be kept relatively stable in polluted water and soil, the tolerance of the flora to adverse environmental conditions is improved, and competition with other microorganisms is avoided, so that BuX and BHA in the water and soil can be degraded more efficiently, and the degradation rate of the water and the soil is increased. And adverse effects on aquatic and soil ecosystems are weakened, and agricultural safety production is guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of pollution remediation, and more specifically, to an immobilized bacterial agent and application thereof in degrading butyl xanthate and benzohydroxamic acid pollution. Background Art

[0002] Flotation is the most effective and widely used method in ore processing. The process inevitably involves the use of flotation reagents. Such reagents cannot be fully used during flotation, and most of them will still remain in the water body. Flotation wastewater is often discharged into the surrounding environment without treatment, causing frequent pollution.

[0003] Among them, butyl xanthate (BuX) and benzohydroxamic acid (BHA) are two typical flotation collectors. These agents separate valuable ores from worthless minerals by adjusting the surface properties of minerals, thereby achieving the purpose of flotation separation.

[0004] In order to improve the recovery rate of the target metal during the flotation process, these agents are often added in excess. However, only part of the agents are consumed during actual use, and the excess will be discharged with the flotation wastewater. Existing wastewater treatment technologies cannot completely remove high concentrations of flotation agents in the water, resulting in high levels of BuX and BHA in the wastewater discharged by the beneficiation plant. High concentrations of BuX and BHA in the water will cause problems such as water odor and increased COD content. Even at extremely low concentration levels, their presence will cause great harm to aquatic organisms, such as affecting the embryonic development of aquatic organisms, causing fish deformities, and inhibiting algae growth. When water bodies contaminated by BuX and BHA are discharged into the environment, they will further pollute surrounding rivers and be used for agricultural irrigation by downstream residents, posing a serious threat to agricultural safety production. BuX and BHA can also affect human health.

[0005] Most of the existing studies have removed them through physical and chemical methods, but considering the cost, environmental protection, renewability and other aspects, the biological remediation method is more economical, effective and eco-friendly. At present, no research has reported the remediation of BuX and BHA composite pollution by microorganisms. In view of the complexity of the actual polluted environment, the degradation effect of degrading bacteria on pollutants is easily affected by adverse environmental conditions and greatly reduced. Therefore, how to improve the bioavailability of BuX and BHA in polluted water by degrading bacteria to improve the remediation effect of functional microorganisms, thereby effectively repairing BuX and BHA composite pollution, is of great significance to ensuring agricultural production safety and human health. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an immobilized bacterial agent and its application in degrading butyl xanthate and benzohydroxamic acid pollution.

[0007] The first object of the present invention is to provide a composition.

[0008] The second object of the present invention is to provide a bacterial agent.

[0009] The third object of the present invention is to provide a method for preparing an immobilized bacterial agent.

[0010] The fourth object of the present invention is to provide an immobilized bacterial agent prepared by any of the preparation methods.

[0011] A fifth object of the present invention is to provide a use of the composition in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or simultaneously degrading butyl xanthate and benzohydroxamic acid.

[0012] The sixth object of the present invention is to provide the application of the bacterial agent or the immobilized bacterial agent

[0013] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0014] The present invention claims a composition comprising Pseudomonas sp. W50 and Cupriavidus sp. HY21.

[0015] The Pseudomonas was deposited in Guangdong Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO.64924;

[0016] The copper-greedy bacteria was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, and its preservation number is: GDMCC No: 65117.

[0017] Preferably, the amount of Pseudomonas and Cupric bacteria is 0.5-2:0.5-2 in volume ratio with an OD 600nm A mixture of 0.8 to 1.2 bacterial suspension.

[0018] More preferably, the amount of Pseudomonas and Cupric bacteria is 1:1 in volume ratio. 600nm A mixture of 1% bacterial suspension.

[0019] The invention also claims to protect a bacterial agent prepared by using the composition.

[0020] And a method for preparing an immobilized bacterial agent, comprising the following steps:

[0021] S1. After the biochar is mixed with the composition according to claim 1, the mixture is fully adsorbed to obtain product 1;

[0022] S2. The sodium alginate aqueous solution is thoroughly mixed with the product 1 to obtain the product 2;

[0023] S3. Product 2 is added dropwise into CaCl2 solution to be fully solidified and obtained after purification.

[0024] Preferably, in product 1, the concentration of biochar is 0.005 to 0.045 g / mL.

[0025] Preferably, in product 1, the concentration of biochar is 0.005 to 0.015 g / mL.

[0026] More preferably, in product 1, the concentration of biochar is 0.01 g / mL.

[0027] Preferably, in product 2, the concentration of sodium alginate is 0.02-0.08 g / mL.

[0028] Preferably, in product 2, the concentration of sodium alginate is 0.03-0.05 g / mL.

[0029] More preferably, in product 2, the concentration of sodium alginate is 0.04 g / mL.

[0030] Preferably, the concentration of the CaCl2 solution is 0.01 to 0.04 g / mL.

[0031] Preferably, the concentration of the CaCl2 solution is 0.015 to 0.025 g / mL.

[0032] More preferably, the concentration of the CaCl2 solution is 0.02 g / mL.

[0033] Preferably, the sufficient curing is curing in a refrigerator at 4 to 8° C. for 12 to 14 hours.

[0034] More preferably, the sufficient curing is curing in a refrigerator at 4°C for 12 hours.

[0035] Preferably, the purification is washing the fixed product with physiological saline.

[0036] Preferably, the biochar preparation method comprises: preparing corn stalk powder, calcining at 450-550° C. for 3-5 hours, washing, making powder, sieving, sterilizing, and cooling.

[0037] More preferably, the washing is performed by sequentially washing with 0.1-0.2 M HCl and pure water.

[0038] More preferably, the sieving is through a 80-100 mesh sieve.

[0039] More preferably, the sterilization is carried out at 121-130° C. for 20-30 min.

[0040] Preferably, the composition is a volume ratio of 0.5-2:0.5-2 of the Pseudomonas OD 600nm The bacterial suspension and the OD of the copper-grasping bacteria were 0.8 to 1.2. 600nm A mixture of 0.8 to 1.2 bacterial suspension.

[0041] More preferably, the method for preparing the bacterial suspension is as follows: the Pseudomonas or Cupriavidus is cultured at 28-37°C for 12-24 hours to obtain activated bacteria; the activated bacteria are cultured in LB liquid culture medium at 150-180r / min, 30-35°C for 12-24 hours; after centrifugation at 3-5°C, 4500-5000r / min for 5-10 minutes, the bacteria are washed with 0.9-1.2% sterile saline and resuspended to prepare the suspension.

[0042] The immobilized bacterial agent prepared by any of the preparation methods also falls within the protection scope of the present invention.

[0043] The use of the composition in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or in simultaneously degrading butyl xanthate and benzohydroxamic acid, also falls within the protection scope of the present invention.

[0044] The application of the bacterial agent or the immobilized bacterial agent is one or more of the following:

[0045] Preparation of products for simultaneous degradation of butyl xanthate and benzohydroxamic acid;

[0046] Application in simultaneous degradation of butyl xanthate and benzohydroxamic acid;

[0047] Application of butyl xanthate and benzohydroxamic acid in remediation of contaminated soils.

[0048] Application of butyl xanthate and benzohydroxamic acid in remediation of polluted water bodies.

[0049] Preferably, the enzymatic activity of soil catalase and / or urease is increased.

[0050] Preferably, the soil is enhanced to increase the abundance of bacterial flora.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The immobilized bacterial agent of the present invention can keep the embedded Pseudomonas and Cupricobacterium flora relatively stable in polluted water and soil, improve their tolerance to adverse environmental conditions, avoid competition with other microorganisms, and thus more efficiently degrade BuX and BHA in water and soil, weaken the adverse effects on aquatic and soil ecosystems, and ensure agricultural safety production.

[0053] (2) When the concentrations of BuX and BHA in the composite polluted water were as high as 100 mg / L, by adding the composite bacterial agent of the present invention to the water, BuX and BHA in the water could be completely removed in only 9 and 30 h, respectively, which was significantly higher than the treatment group using free bacteria alone.

[0054] (3) The composite bacterial agent capable of degrading BuX and BHA of the present invention has a wide range of applications and can effectively reduce the content of BuX and BHA in water bodies contaminated by BuX and BHA under extreme pH, temperature or heavy metal stress conditions, thereby reducing the harm to the water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The colony morphology of the strains on the nutrient agar plate; A is strain W50; B is strain HY21.

[0056] Figure 2 Shown are the morphological characteristics of the strains under SEM; A is strain W50; B is strain HY21.

[0057] Figure 3 This is the electrophoresis diagram of PCR amplification products. M: DNA molecular weight standard; 1: W50 amplification product; 2: HY21 amplification product.

[0058] Figure 4 This is the phylogenetic tree of strain W50 based on 16S rDNA sequence.

[0059] Figure 5 This is the phylogenetic tree of strain HY21 based on 16S rDNA sequence.

[0060] Figure 6 The chromatograms of quantitative ion (A) and qualitative ion (B) of BHA.

[0061] Figure 7 The degradation kinetic curve (A) and fitting curve (B) of strain HY21 for different initial concentrations of BHA.

[0062] Figure 8 is the tolerance of strain W50 to different initial BuX concentrations.

[0063] Fig. 9 This is the antagonism between strain W50 and strain HY21.

[0064] Fig.10 Degradation of BuX (A) and BHA (B) by mixed bacterial flora FB in single and combined pollution systems;

[0065] Fig.11 This is the SEM image of biochar.

[0066] Fig.12 FT-IR image of biochar.

[0067] Fig.13 The appearance (A) and particle size (B) of the immobilized bacterial agent.

[0068] Fig.14 The surface morphology and internal structure of the immobilized bacteria; A and B are the surface morphology; C and D are the internal structure.

[0069] Fig.15 FT-IR images of biochar, SB and IB.

[0070] Fig.16 Degradation effects of each treatment group on BuX and BHA; A is the degradation effect on BuX; B is the degradation effect on BHA.

[0071] Fig.17 These are immobilized bacterial agents prepared with different sodium alginate contents; the sodium alginate contents in A to D are 1%, 2%, 3% and 4% respectively.

[0072] Fig.18 The degradation effect of immobilized bacteria with different sodium alginate contents on BHA and BuX.

[0073] Fig.19 The degradation effects of immobilized bacteria with different biochar contents on BHA and BuX.

[0074] Fig. 20 The degradation effect of immobilized bacteria at different CaCl2 concentrations on BHA and BuX

[0075] Fig.21 Degradation ability of immobilized bacteria under strong acid and strong base; degradation of BuX (A) and BHA (B) by each treatment group under strong acid condition (pH=5); degradation of BuX (C) and BHA (D) by each treatment group under strong alkaline condition (pH=9).

[0076] Fig. 22 Degradation ability of immobilized bacteria at high and low temperatures; degradation of BuX (A) and BHA (B) by each treatment group under high temperature conditions (40°C); degradation of BuX (C) and BHA (D) by each treatment group under low temperature conditions (20°C).

[0077] Fig.23 Cd 2+ Degradation of BuX (A) and BHA (B) by each treatment group at a concentration of 5 mg / L.

[0078] Fig.24 The degradation of BuX (A) and BHA (B) by immobilized bacteria after 5 cycles.

[0079] Fig.25 The degradation effect of immobilized bacteria on BuX and BHA after storage for different days.

[0080] Fig.26 The changes of soil BuX concentration in each treatment group at different restoration periods; A is group S1; B is group S2.

[0081] Fig. 27 The changes in soil pH in each treatment group during different restoration periods; A is group S1; B is group S2.

[0082] Fig.28 Changes in soil catalase activity in each treatment group at different restoration periods; A is group S1; B is group S2.

[0083] Fig.29 Changes in soil urease activity in each treatment group at different restoration periods; A is group S1; B is group S2.

[0084] Fig.30 These are principal coordinate analysis diagrams of each treatment group; A is group S1; B is group S2.

[0085] Fig.31 is the relative abundance of soil bacterial communities at the phylum level in each treatment group.

[0086] Fig.32 is the relative abundance of soil bacterial communities at the genus level in each treatment group

[0087] Fig.33 is the relative abundance of Pseudomonas in each treatment group. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0089] Butyl xanthate (BuX) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Aladdin) with a purity of >95%; Benzohydroxamic acid (BHA) was purchased from Sigma-Aldrich, USA with a purity of >99%; Chromatographic grade methanol was purchased from Sigma-Aldrich, USA; Hydrochloric acid was purchased from Guangzhou Chemical Reagent Co., Ltd. with analytical purity; The remaining reagents were all analytically pure and purchased from Tianjin Damao Chemical Reagent Co., Ltd.

[0090] Liquid basal salt medium (MSM): K2HPO4 1.5g / L, KH2PO4 0.5g / L, (NH4)2SO4 0.5g / L, MgSO4·7H2O 0.2g / L and NaCl 1.0g / L, pH value is about 7.0.

[0091] LB broth medium: peptone 10 g / L, yeast extract 5 g / L and sodium chloride 5 g / L, pH value is about 7.0.

[0092] Nutrient agar medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, agar 15g / L, pH value is about 7.0.

[0093] The soil samples used to isolate degrading bacteria in this study were taken from the Dabaoshan mining area in Shaoguan, Guangzhou.

[0094] Example 1 Enrichment and separation and purification of strains

[0095] 1. Experimental Methods

[0096] 100 mL of MSM medium was sterilized in a 250 mL conical flask. After cooling, BuX or BHA was added to the flask to make the initial concentration reach 10 mg / L. 5 g of contaminated soil collected from the mining area (Dabaoshan mining area, Shaoguan, Guangzhou) was added to the flask and cultured in a shaker at 35°C and 150 rpm in the dark for 7 days. On the 7th day of culture, the inoculation amount was transferred to a new MSM medium at 10%, and the concentration of pollutants was gradually increased for acclimatization, and the transfer was continuous for 5 times.

[0097] Take the last culture solution and evenly spread it on the nutrient agar plate. After constant temperature culture for 48 hours, pick the colony and repeatedly streak it until a single colony is obtained. Inoculate the single bacteria into MSM medium with 100 mg / L BHA or BuX, culture it at 35°C and 150 rpm for 72 hours, test its degradation effect, and inoculate the strain into the test tube slant culture medium, and store it in a refrigerator at 4°C for use.

[0098] 2. Experimental Results

[0099] After gradient acclimation, a strain that can use BuX as the sole carbon source was selected and named W50, and another strain that can use BHA as the sole carbon source was named HY21. The two strains can completely degrade BuX or BHA with an initial concentration of 100 mg / L in a short time.

[0100] Example 2 Identification of strain morphological characteristics

[0101] 1. Observe the characteristics of colonies

[0102] 1. Experimental methods

[0103] The strains W50 and HY21 screened in Example 1 were inoculated onto nutrient agar plates, respectively, and inverted in an incubator at 35° C. for overnight culture. The shape, size, transparency, edge, color, etc. of the strains were observed.

[0104] 2. Experimental results

[0105] After overnight culture, the colony morphology of the two strains was observed. On the nutrient agar plate, strain W50 was round, light yellow, translucent, smooth in surface, convex in the middle, and sticky in texture ( Figure 1 A in the figure); strain HY21 showed milky white, irregular, opaque, and rough surface ( Figure 1 B).

[0106] 2. SEM observation of bacterial morphology

[0107] 1. Experimental methods

[0108] The strains W50 and HY21 were inoculated in LB broth culture medium, and 800 μL of the bacterial solution after 24 hours of culture was taken into a 1.5 mL centrifuge tube, centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded. The obtained bacterial precipitate was washed with sterile saline, centrifuged and the supernatant was discarded, and this operation was repeated 3 times. 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to the washed bacteria, vortexed and mixed, and allowed to stand overnight in a 4 ° C refrigerator. After fixation, centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the bacteria were collected and washed with sterile saline 3 times. Then, gradient dehydration was performed, that is, 30%, 50%, 70%, 80% and 90% ethanol were added once, and 100% ethanol was added twice. Each dehydration treatment required standing for 15 minutes. After the last dehydration treatment, the bacteria were dropped on a cover glass and placed in a freeze dryer for freeze drying. The dried samples were sprayed with gold and then observed on the machine.

[0109] 2. Experimental results

[0110] SEM observation showed that strain W50 was long rod-shaped (1.5-3.0×0.5 μm) with a relatively smooth surface ( Figure 2 A in the figure); strain HY21 is short rod-shaped (0.8-2.0×0.5μm) with a rough surface ( Figure 2 B).

[0111] 3. Physiological and biochemical identification of strains

[0112] 1. Experimental methods

[0113] The physiological and biochemical experiments of strains W50 and HY21 were carried out with reference to the Manual of Common Bacterial Identification and Bergey's Manual of Determinative Bacteriology.

[0114] 2. Experimental results

[0115] Gram staining results showed that both strains were Gram-negative bacteria, with the strain W50 methyl red test, nitrate reduction, starch hydrolysis, urease production, hydrogen sulfide, and acetylmethylcarbinol tests all being negative, and the catalase, oxidation reaction, ornithine decarboxylase, and lysine decarboxylase tests being positive, and the strain was unable to utilize malonate, lactose, and glucose ( Table 1 );

[0116] Table 1 Physiological and biochemical characteristics of strain W50

[0117]

[0118] Note: “+” indicates that it can be used or is positive; “-” indicates that it cannot be used or is negative.

[0119] The methyl red test, nitrate reduction, catalase, starch hydrolysis, hydrogen sulfide, and acetylmethylcarbinol tests of strain HY21 were all negative, while the oxidation reaction, ornithine decarboxylase, lysine decarboxylase test, and urease production were positive. The strain could not utilize lactose and glucose, but could utilize malonate (Table 2).

[0120] Table 2 Physiological and biochemical characteristics of strain HY21

[0121]

[0122] 4. Molecular Biological Identification of Strains

[0123] 1. Experimental methods

[0124] PCR amplification: The 16S rDNA fragments of strains W50 and HY21 were amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively.

[0125] PCR reaction system: Taq Mixture, 12.5 μL; ddH2O, 9.5 μL; 27F, 1 μL; 1492R, 1 μL; DNA template, 1 μL. The total reaction system is 25 μL.

[0126] PCR reaction conditions: 95℃, initial preheating for 5min; melting at 94℃, 45s, annealing at 55℃, 45s, extension at 72℃, 1min15s, 32 cycles, and finally 72℃ for 10min. After PCR amplification, the size and specificity of the amplified fragments were detected by 1.5% agarose gel electrophoresis, and photographed in a gel imaging system. The PCR amplification products were purified, recovered and sequenced by Shanghai Bioengineering Company.

[0127] Construction of phylogenetic tree: The 16S rDNA sequences of the two strains were obtained by sequencing by Sangon Company and submitted to the GenBank database. BLAST was used for comparison analysis, and ClustalX software was used for homology analysis of related sequences with high matching degree. Neighborhood-joining method in MEGA 11.0 software was used for relationship analysis to construct the phylogenetic tree of the strains.

[0128] 2. Experimental results

[0129] The genomic DNA of strain W50 and strain HY21 were used as templates for PCR amplification using 16S rDNA bacterial universal primers. The electrophoresis results of the amplified products are shown in Figure 3 The amplified products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for purification and sequencing, and the 16S rDNA sequencing results were obtained. The 16S rDNA sequencing results of strain W50 and strain HY21 were submitted to the NCBI Genbank database for BLAST comparison. The sequences with high homology to the target strain in the BlAST comparison analysis results were used for multiple sequence alignment, and the phylogenetic trees of strain W50 and strain HY21 were constructed by the neighbor-joining method ( Figure 4 and Figure 5 ).

[0130] The phylogenetic tree results showed that the 16S rDNA of strain W50 had the highest homology with Pseudomonas juntendi BML3NR180457 (Genbank accession number: PP737842), and also had a high homology with other strains of the genus (Pseudomonas). Combined with the physiological and biochemical identification results of the above strain W50, it was preliminarily determined that strain W50 belonged to the genus (Pseudomonas); the 16S rDNA of strain HY21 had the highest homology with Cupriavidus basilensis strain N1129 (MN691130.1), and also had a high homology with other strains of the genus (Cupriavidus). Combined with the physiological and biochemical identification results of strain HY21, it was preliminarily determined that strain HY21 belonged to the genus (Cupriavidus).

[0131] Strain W50 and strain HY21 were preserved separately: strain W50 was named Pseudomonas sp. W50, and was preserved in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the preservation number: GDMCC NO.64924, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; strain HY21 was named Cupriavidus sp. HY21, and was preserved in Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, with the preservation number: GDMCC No: 65117, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0132] Example 3 Degradation effect of strain HY21 on BHA

[0133] 1. Experimental Methods

[0134] At pH 5.5, temperature 30.5°C, inoculum 1.0 (OD 600 , 2% volume fraction) degradation conditions, the bacterial suspension was inoculated into 50mL MSM medium. The BHA concentration in the culture medium was set to 50, 100, 150, and 200 mg / L, respectively, and it was placed in a constant temperature oscillating box with 150rpm shaking culture for 48h. Samples were taken when it was degraded to 0, 6, 12, 24, 30, 36, and 48h, and the concentration of BHA remaining in the culture solution was determined. The degradation kinetic curve of strain HY21 for different initial concentrations of BHA was drawn. The control group was not inoculated, and three parallels were set for each treatment group.

[0135] The method for detecting the concentration of BHA is:

[0136] 1. Sample pretreatment method

[0137] 1 mL of the culture medium to be tested was placed in a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 min. 100 μL of the supernatant was diluted 100 times with methanol-water (50 / 50, v / v) as the solvent, and finally filtered through a 0.22 μm microporous membrane, and the filtrate was collected into a sample injection bottle, and the BHA content was determined by LC-MS / MS.

[0138] 2. LC-MS / MS detection method of BHA

[0139] An LC-MS / MS detection method was established using AB Sciex 5500MD high performance liquid chromatography-tandem mass spectrometer.

[0140] Chromatographic conditions: the chromatographic column is Water Xbridge C18 (3.0mm×50mm, 5μm); mobile phase A is water; mobile phase B is methanol; column flow rate is 0.40mL / min; column temperature is 40℃; injection volume is 5μL; mobile phase gradient elution program is as shown in Table 3.

[0141] Table 3 Mobile phase gradient elution program

[0142]

[0143] Mass spectrometry conditions:

[0144] Ion source: electrospray ion source negative ion (ESI-); electrospray voltage: -4500V; nebulizer gas temperature: 500°C; nebulizer gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 35psi; collider pressure: 6psi; scanning mode: multiple reaction monitoring mode (MRM). The mass spectrometry parameters of BHA are shown in Table 4, and the quantitative ion and qualitative ion chromatograms are shown in Figure 4 shown.

[0145] Table 4

[0146]

[0147] 3. BHA standard curve

[0148] Take 10 μL of BHA standard solution (1000 mg / L) and add it to the injection bottle, dilute it with 990 μL of chromatographic grade methanol, and obtain a BHA standard stock solution with a concentration of 10 mg / L. Prepare solutions of 50, 75, 150, 300, 600, and 1200 ng / mL according to the gradient dilution method, and the solvent is methanol-water (50 / 50, v / v) solution. According to the LC-MS / MS detection method of BHA above, the peak area corresponding to each concentration is determined, and the peak area determined is Y, and the corresponding standard solution concentration is X, and the standard curve "Y=a+bX" is drawn.

[0149] 2. Experimental Results

[0150] The chromatograms of the quantitative ion (A) and the qualitative ion (B) of BHA are shown in Figure 6 .

[0151] The results are as follows Figure 7As shown in Figure A, the results show that strain HY21 can tolerate a BHA concentration range of 50-200 mg / L. A strain of Klebsiella BHA-degrading bacteria has been reported, and the degradation rate of the initial concentration of 100 mg / L BHA is only 85.04% after 12 days of cultivation (Hu Chun, Wang Can, Gong Wenqi, et al. Study on the microbial degradation of three hydroxamic acid collectors [J]. Hubei Agricultural Science, 2013, 52(11): 2505-2507). In comparison, strain HY21 can tolerate a higher concentration of BHA and has a higher degradation efficiency for BHA.

[0152] Example 4 Degradation effect of strain W50 on BuX

[0153] 1. Experimental Methods

[0154] BuX was added to 50 mL of sterilized MSM medium to make its initial concentration reach 100, 300, 500, 700 and 1000 mg / L respectively. The initial pH of the system was 7.0. 2% W50 bacterial suspension (OD 600 =1.0), and then placed in a constant temperature oscillator at 35°C and 150 rpm in the dark for 12 hours. Samples were taken every 2 hours to determine the BuX concentration, and the uninoculated treatment was used as a blank control. Three parallels were set for each group.

[0155] The method for detecting the concentration of BuX is:

[0156] 1. Sample pretreatment method

[0157] Take 1 mL of culture medium into a 1.5 mL centrifuge tube and centrifuge at 8000 rpm for 5 min. After centrifugation, take 300 μL of supernatant and dilute 10 times to obtain the test solution, and immediately measure the BuX content by UV-Vis (to prevent BuX degradation).

[0158] 2. Detection method of BuX

[0159] The absorbance value of the test solution at 301 nm can be measured and the BuX content in the solution can be calculated based on the standard curve.

[0160] 3. BuX standard curve

[0161] Accurately weigh a certain amount of BuX, dissolve it in pure water and dilute it to 50 mL to obtain a BuX standard stock solution with a final concentration of 1000 mg / L. Dilute the standard stock solution step by step to prepare standard solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 25.0 mg / L, respectively. By measuring the absorbance of the standard solution of each gradient at 301 nm, with it as Y and the corresponding solution concentration as X, a standard curve "Y=a+bX" is prepared.

[0162] 2. Experimental Results

[0163] The results show Figure 8 The results showed that strain W50 could completely degrade BuX within 12 hours when the initial concentration was 100-1000 mg / L, indicating that strain W50 could tolerate extremely high concentrations of BuX and there was no obvious hysteresis effect in the degradation process. BuX is often added excessively during the flotation process, resulting in a high content of BuX in the discharged flotation wastewater. Therefore, it is necessary to screen strains that can tolerate high concentrations of BuX, and strain HY21 has excellent application prospects in high-concentration BuX wastewater.

[0164] Example 5 Construction of mixed bacterial flora

[0165] 1. Experimental Methods

[0166] The strain W50 and strain HY21 isolated in Example 1 were streaked on a nutrient agar medium by the cross method, and the medium was inverted and placed in a 35° C. constant temperature incubator for 24 hours. The growth of the strains at the intersection was observed to see if an obvious inhibition zone appeared.

[0167] 2. Experimental Results

[0168] The results are as follows Fig. 9 As shown, the results of cross-streaking of strain W50 and strain HY21 showed that the strains at the intersection grew well, and there was no growth inhibition between the two strains. Therefore, strain W50 and strain HY21 can be used to construct a bacterial community.

[0169] Strain W50 and strain HY21 were inoculated into LB medium and cultured overnight (150-180 rpm, 30-35°C for 12-24 h). When the strains grew to the logarithmic phase, they were centrifuged at 4500 rpm for 10 min to obtain bacterial precipitates. The bacterial precipitates were washed three times with sterile physiological washing, resuspended with physiological saline and the OD was adjusted. 600 to 1.0, and then mixed at a ratio of 1:1 (V / V) to obtain a mixed bacterial community (i.e., free bacterial community, FB).

[0170] Example 6 Degradation effect of bacterial flora in single or composite pollution system

[0171] 1. Experimental Methods

[0172] 50 mL of MSM culture medium containing only 100 mg / L BuX or BHA and both were prepared respectively, and the mixed bacterial community (i.e., isolated bacterial community, FB) obtained in Example 5 was inoculated at a volume ratio of 2%, and the conical flask was placed in a constant temperature condition of 35° C. and 150 rpm for shaking culture for 48 h. Samples were taken at regular intervals to determine the contents of remaining BuX and BHA in the solution at different time points (the method was the same as in Examples 3 and 4).

[0173] 2. Experimental Results

[0174] The results are as follows Fig.10 The results showed that it took 9 h and 30 h, respectively, for the mixed bacterial community in a single pollution system to completely degrade BuX or BHA with an initial concentration of 100 mg / L, while the time required for complete degradation of BuX and BHA in the composite system was prolonged to 24 h and 48 h, respectively.

[0175] Example 7 Preparation and Characterization of Biochar

[0176] 1. Preparation of Biochar

[0177] The washed and dried corn stalks were crushed and prepared into powder, which was placed in a crucible, covered with a crucible lid, placed in a 500°C muffle furnace, and calcined at high temperature for 4 hours under a nitrogen atmosphere. The biochar obtained after calcination was also washed with 0.1M HCl for demineralization, and then washed with deionized water to completely remove the residual acid and ash on the surface of the biochar. Finally, the biochar was ground into fine powder, passed through a 100-mesh sieve, sterilized at 121-130°C for 20-30 minutes, cooled to room temperature, and placed in a dry environment for use.

[0178] 2. Scanning the surface of biochar using SEM

[0179] 1. Experimental methods

[0180] The biochar was washed with sterile saline, the supernatant was discarded, and this operation was repeated 3 times. 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to the washed bacterial agent, vortexed and mixed, and allowed to stand in a 4°C refrigerator for 20 minutes. After fixation, the supernatant was discarded, the bacterial agent was collected and washed 3 times with sterile saline. Then, gradient dehydration was performed, that is, 30%, 50%, 70%, 80%, 90% (volume fraction) ethanol was added once, and 100% (volume fraction) ethanol was added twice. Each dehydration treatment required standing for 15 minutes. After the last dehydration treatment, the bacteria were dropped on a cover glass and placed in a freeze dryer for freeze drying. The dried samples were sprayed with gold and then observed on the machine.

[0181] 2. Experimental results

[0182] Through SEM results ( Fig.11 ) It can be seen that the surface of biochar presents a porous structure with large pores and no obvious collapse. This structural feature can provide a suitable growth environment for microorganisms, promote the colonization of microorganisms on its surface, and also increase the specific surface area of ​​biochar, providing more active sites for pollutant adsorption.

[0183] 3. FT-IR scanning of biochar

[0184] 1. Experimental methods

[0185] After the biochar is fully freeze-dried, add an appropriate amount of potassium bromide powder, grind and mix, press into tablets, and place them in a Fourier infrared spectrometer to scan in the range of 4000 to 400 cm-1.

[0186] 2. Experimental results

[0187] The FT-IR results of biochar are shown in Figure 12. In the FT-IR graph, infrared absorption peaks of various functional groups were observed, such as 3428 cm -1 (-OH), 1576cm -1 (-C=O), 1384cm -1 (-COOH), and 1081cm -1 (-CO), the above results indicate that the prepared biochar contains abundant oxygen-containing functional groups, which increases the active sites for biochar to adsorb pollutants and can provide nutrients for microorganisms to promote their growth and reproduction.

[0188] Example 8 Preparation of immobilized bacterial agent and degradation effect

[0189] 1. Preparation method

[0190] The preparation of immobilized bacteria (IB) adopts the adsorption-embedding method, which mainly includes adsorption, mixing and embedding, three steps:

[0191] Adsorption: Weigh 0.5 g of the biochar prepared in Example 7 and put it into a conical flask. After sterilization, wait for it to cool down. Pour 50 mL of the mixed bacterial community FB (obtained in Example 5) into the biochar and shake it in a shaker at 35°C for 2 h to perform adsorption as an adsorption system;

[0192] Mixing: Weigh 2g of sodium alginate and dissolve it in 50mL of water, heat and stir until it is completely dissolved, and then sterilize at 121℃ for 20min. After cooling to room temperature, pour the adsorbed adsorption system into the sodium alginate solution, and place the mixed solution on a magnetic stirrer and stir for 30min to fully mix it;

[0193] Embedding: The mixed solution was dripped into a 0.02 g / mL CaCl2 solution drop by drop at a uniform speed using a syringe to embed the immobilized beads. After embedding, the immobilized beads were placed in a 4°C refrigerator to continue solidification for 12 hours. After 12 hours, the immobilized beads were washed 3 times with sterile saline to remove excess CaCl2 on the surface; the immobilized beads were subsequently stored in a 4°C refrigerator to obtain an immobilized bacterial agent.

[0194] At the same time, immobilized beads (SB) without added bacteria were prepared: sterile physiological saline was directly added to the biochar during adsorption (instead of the mixed bacterial community FB obtained in Example 5), and the remaining steps were consistent with the preparation process of the immobilized bacterial agent.

[0195] 2. Apparent properties of immobilized bacteria (IB)

[0196] 1. Experimental methods

[0197] (1) Observe the shape of the inoculum with the naked eye, take a portion of the inoculum and weigh it, and use a vernier caliper to measure the diameter of the inoculum.

[0198] (2) Determination of mass transfer efficiency: Add 10% of the pellets to 10 mL of 0.01% methylene blue solution, place in a constant temperature oscillator at 30°C and 150 rpm for 24 h, and observe the change in the absorbance of the solution at 665 nm before and after the addition of the immobilized bacterial agent.

[0199] (3) Mechanical strength measurement: Add 10 immobilized bacteria of uniform size into 20 mL of pure water, oscillate in an incubator at 220 rpm / min for 5 days, and observe the rupture of the bacteria.

[0200] 2. Experimental results

[0201] Finished products of immobilized bacteria Fig.13 As shown, its apparent properties are shown in Table 5. The results showed that the immobilized bacterial agent was regular spherical and had high mass transfer efficiency and mechanical strength.

[0202] Table 5 Apparent properties of immobilized bacterial agents

[0203]

[0204] 3. SEM observation of the surface morphology and internal structure of immobilized bacteria (IB)

[0205] 1. Experimental methods

[0206] The immobilized bacterial agent (IB) was washed with sterile saline, the supernatant was discarded, and this operation was repeated 3 times. 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to the washed bacterial agent, vortexed and mixed, and allowed to stand in a 4°C refrigerator for 20 minutes. After fixation, the supernatant was discarded, the bacterial agent was collected and washed 3 times with sterile saline. Then, gradient dehydration was performed, that is, 30%, 50%, 70%, 80%, 90% (volume fraction) ethanol was added once, and 100% (volume fraction) ethanol was added twice. Each dehydration treatment required standing for 15 minutes. After the last dehydration treatment, the bacteria were dropped on a cover glass and placed in a freeze dryer for freeze drying. The dried samples were sprayed with gold and then observed on the machine to observe their surface morphology and internal structure.

[0207] 2. Experimental results

[0208] The surface morphology of the immobilized bacteria is as follows Fig.14 As shown, Figure A shows the overall morphology of the immobilized bacteria, which presents a regular spherical shape; Figure B is the surface morphology of the immobilized bacteria. Since the spheres shrink after being dehydrated by ethanol gradient, wrinkles appear on the surface, but a large number of strains can still be observed to be embedded in them; Figures C and D are scans of the cross-section of the immobilized bacteria to observe its internal structure; in Figure C, it can be seen that there are a large number of honeycomb holes inside the spheres. The existence of this structure can increase the porosity and specific surface area of ​​the carrier, reduce diffusion resistance, and is conducive to the transmission of oxygen and nutrients, providing a better microenvironment for the growth and metabolism of microorganisms, thereby accelerating their degradation of pollutants; in Figure D, a large number of bacteria were observed to be attached to the pores inside the carrier, indicating that the embedded bacteria grew well in the immobilized carrier.

[0209] 4. FT-IR analysis of immobilized bacteria (IB)

[0210] 1. Experimental methods

[0211] After the immobilized beads (SB) and immobilized bacterial agent (IB) without bacterial flora were fully freeze-dried, they were ground and mixed with appropriate amount of potassium bromide powder, pressed into tablets and placed in a Fourier infrared spectrometer. 1 The FT-IR spectrum was obtained by scanning in the range to analyze the composition of its functional groups.

[0212] 2. Experimental results

[0213] According to the FT-IR results ( Fig.15 ) as shown, 3428cm -1 The absorption peak at 2972cm is generated by OH stretching vibration. The absorption intensity of this peak is enhanced after the biochar is combined with sodium alginate, indicating that a large number of hydrogen bonds are generated between the -OH groups of biochar and sodium alginate. -1 The new peak at 1418 cm in SB is attributed to the stretching vibration of CH, indicating that the biochar is firmly combined with the sodium alginate matrix due to the tight connection formed by hydrogen bonds. -1 、1618cm -1 The absorption peaks at 1081cm are characteristic peaks of sodium alginate, representing the symmetric and antisymmetric stretching vibrations of COO-, respectively. -1 and 1034cm -1 This corresponds to the deformation of -CO and the vibration of -COC in sodium alginate. Compared with IB, the functional groups of SB are all present in IB, and the intensity of the absorption peak is significantly enhanced, which is closely related to the generation of hydrogen bonds during the material composite process, indicating that the materials are well combined.

[0214] 5. Degradation effect of immobilized bacteria (IB) on BuX and BHA

[0215] 1. Experimental methods

[0216] Take 2g of the prepared immobilized bacterial agent (IB) and 2g of the immobilized beads (SB) without embedded bacteria, and 1mL of the free bacterial colony (FB) prepared in Example 5, add them to 50mL of MSM medium, the concentration of BuX and BHA in the medium is 100mg / L, and place it in a shaking table at 35°C and 150rpm for 24h. Samples were taken when the culture was 12h and 24h, and the contents of BuX and BHA were determined respectively (the method is the same as that of Examples 3 and 4), and three parallels were set for each group, and the control group was an MSM medium without inoculation treatment and containing only 100mg / L BuX and BHA.

[0217] 2. Experimental results

[0218] The results are as follows Fig.16 As shown in the figure, the degradation rate of pollutants is ranked from fast to slow as follows: IB>FB>SB. In the IB group, 100 mg / L BuX and BHA can be completely degraded in only 9 hours and 30 hours, while the FB group needs 24 hours and 48 hours to completely degrade BuX and BHA respectively. The removal rates of the SB group in 48 hours are only 50.45% and 22.25%, respectively. The results show that the preparation of immobilized bacterial agents significantly improves the degradation rate of BuX and BHA by the bacterial community in the complex pollution system. The concentration of pollutants in the SB group is lower than that in the CK group, which also shows that the immobilized material has a certain adsorption effect on the pollutants. Comparison between the IB and SB treatment groups shows that the main reason for the decrease in pollutant concentration in IB is the degradation effect of the strain.

[0219] Example 9 Effect of Sodium Alginate Content on the Degradation of BuX and BHA by Immobilized Bacteria

[0220] 1. Experimental Methods

[0221] The immobilized bacterial agent (IB) was prepared according to the method of Example 1. The specific method is as follows:

[0222] Adsorption: same as Example 8;

[0223] Mixing: Weigh 1g (i.e. 1%), 2g (i.e. 2%), 3g (i.e. 3%) and 4g (i.e. 4%) of sodium alginate and dissolve them in 50mL of water. Heat and stir until they are completely dissolved and sterilize at 121℃ for 20min. After cooling to room temperature, pour the adsorbed system into the sodium alginate solution and stir the mixed solution on a magnetic stirrer for 30min to mix it thoroughly.

[0224] Embedding: Same as Example 8.

[0225] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0226] 2. Experimental Results

[0227] The apparent morphology of the immobilized bacterial agents prepared at different sodium alginate contents is as follows: Fig.17 As shown, the immobilized beads prepared when the sodium alginate content is 2%, 3%, and 4% are all regular spheres, but the increase in sodium alginate content will lead to an increase in the viscosity of the material, and the difficulty of its preparation will also increase accordingly. When the sodium alginate content is 1%, the beads are irregular in shape and are very easy to break during use.

[0228] according to Fig.18 The results of the degradation of BuX and BHA show that when the sodium alginate content is 1% and 2%, the degradation rate of BuX is higher than 90% after 12h of culture, and more than 80% of BHA can be degraded after 24h of reaction. When the sodium alginate content reaches 3% and 4%, the degradation rate of BuX decreases slightly, to 88.62% and 88.99% respectively, and the degradation rate of BHA decreases significantly to 53.02% and 49.14%. This shows that the increase of sodium alginate content will lead to the deterioration of the degradation effect of the immobilized bacteria on the two pollutants, and the reason may be that the increase of sodium alginate content causes the mass transfer efficiency of the beads to decrease.

[0229] Therefore, the immobilized bacterial agent prepared with 2% sodium alginate has the best apparent morphology and the best effect on pollutant degradation.

[0230] Example 9 Effect of biochar content on the degradation of BuX and BHA by immobilized bacteria

[0231] 1. Experimental Methods

[0232] The immobilized bacterial agent (IB) was prepared according to the method of Example 8. The specific method is as follows:

[0233] Adsorption: 0.5 g (i.e., content 0.5%), 1 g (i.e., content 1%), 1.5 g (i.e., content 1.5%), and 2 g (i.e., content 2%) of the biochar prepared in Example 7 were weighed and placed into a conical flask, sterilized and cooled, 50 mL of the mixed bacterial community FB (obtained in Example 5) was poured into the biochar, and the conical flask was placed in a shaker at 35°C for 2 h for adsorption as an adsorption system;

[0234] Mixing: Same as Example 8;

[0235] Embedding: Same as Example 8.

[0236] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0237] 2. Experimental Results

[0238] There is no obvious difference in the morphology of immobilized bacteria with different biochar contents.

[0239] from Fig.19 It can be seen that when the biochar content is 0.5%, the degradation rates of BuX and BHA are both the highest, 97.23% and 96.32% respectively. When the biochar content exceeds 0.5%, the degradation rate of the pollutants decreases significantly.

[0240] Example 9 Effect of CaCl2 concentration on the degradation of BuX and BHA by immobilized bacteria

[0241] 1. Experimental Methods

[0242] The immobilized bacterial agent (IB) was prepared according to the method of Example 8. The specific method is as follows:

[0243] Adsorption: same as Example 8;

[0244] Mixing: Same as Example 8;

[0245] Embedding: The mixed solution was dripped into 0.02g / mL, 0.03g / mL, 0.04g / mL and 0.05g / mL CaCl2 solutions at a uniform speed through a 50mL syringe to embed the immobilized beads. After embedding, the immobilized beads were placed in a 4°C refrigerator to continue solidification for 12 hours. After 12 hours, the immobilized beads were washed 3 times with sterile saline to remove excess CaCl2 on the surface; the immobilized beads were subsequently stored in a 4°C refrigerator to obtain an immobilized bacterial agent.

[0246] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0247] 2. Experimental Results

[0248] The results are as follows Fig. 20 As shown in the figure, the CaCl2 concentration has little effect on the degradation of BuX, but it will significantly affect the degradation of BHA. With the increase of CaCl2 concentration, the degradation rates of BuX are 94.28%, 96.05%, 95.87% and 85.02%, and the degradation rates of BHA are 97.63%, 55.02%, 83.37% and 47.56%, respectively. Considering the degradation effects of the two pollutants, the CaCl2 concentration of 2g / ml is the most appropriate.

[0249] Example 10 Degradation ability of immobilized bacterial agent under strong acid and strong base

[0250] 1. Experimental Methods

[0251] The pH of the MSM culture medium was accurately adjusted to 5 and 9 using a pH meter, and BuX and BHA were added to the packaged MSM culture medium to make the concentrations of the two pollutants reach 100 mg / L.

[0252] 2% (volume ratio) of the free bacterial community (FB) prepared in Example 5, 2 g of the immobilized bacterial agent (IB) prepared in Example 8, and 2 g of the immobilized beads (SB) prepared in Example 8 without bacteria were added to 50 mL of MSM medium, respectively. After inoculation, the conical flask was placed at a constant temperature of 35° C. and cultured at 150 rpm for 48 h. When the reaction reached different time points, samples were taken to determine the concentrations of BuX and BHA. Three parallels were set for each treatment group, and the blank group was the MSM medium (CK) without inoculation treatment.

[0253] 2. Experimental Results

[0254] like Fig.21 As shown in the figure, IB can achieve efficient degradation of BuX and BHA in strong acid or strong base environment. At pH 5, it only takes 6h and 24h for BuX and BHA in the IB group to be completely degraded; when the pH is increased to 9, the degradation time is 12h and 0h, respectively.

[0255] The FB group can also achieve a relatively efficient degradation of BuX and BHA under strong acid or strong alkaline environments. When the pH is 5, FB can completely degrade BuX and BHA in the composite system in just 9h and 30h. When the pH reaches 9, the degradation rates of BuX and BHA are only 58.21% and 23.22% after culturing for 24h and 48h, respectively.

[0256] In the SB group, pH was 5 or 9, the removal rates of BHA at 48 h were 25.78% and 18.46%, respectively. In comparison, the removal rates of BuX at 24 h were quite different. The BuX in the former was completely removed, while the removal rate of BuX in the latter was only 39.39% at the same time.

[0257] The results show that the immobilized bacterial agent prepared by the adsorption-encapsulation method has a significantly improved removal ability for BuX and BHA in an adverse pH environment under the protection of the biochar-sodium alginate carrier.

[0258] Example 11 Degradation ability of immobilized bacterial agents at high and low temperatures

[0259] 1. Experimental Methods

[0260] 2% (volume ratio) of the free bacterial group (FB) prepared in Example 5, 2 g of the immobilized bacterial agent (IB) prepared in Example 8, and 2 g of the immobilized beads (SB) without bacteria prepared in Example 8 were added to 50 mL of MSM culture medium. The blank control (CK) was not inoculated with bacteria. The initial pH of the degradation system was 7. The temperatures of the constant temperature shaker were set to 20° C. and 40° C., respectively. The conical flask was then placed therein, and the reaction was carried out at a speed of 150 rpm for 48 h. Samples were taken at regular intervals to observe the changes in the concentrations of BuX and BHA in the solution.

[0261] 2. Experimental Results

[0262] Depend on Fig. 22 It can be seen that no matter under high or low temperature conditions, the IB group was the first to degrade all BuX and BHA.

[0263] IB only needs 30 hours to degrade BHA at 40℃ or 20℃; at 40℃, IB can completely degrade BuX in 6 hours. Although high temperature can accelerate the decomposition of BuX, compared with CK, it can be seen that the degradation of BuX by the strain is still the main factor in this process. When the temperature drops to 20℃, the low temperature will inhibit the decomposition of BuX, but IB can still reach the reaction endpoint within 9 hours.

[0264] At 40℃, when FB was degraded for 24h and 8h, the degradation rates of BuX and BHA were 96.61% and 70.35% respectively. However, at 20℃, the degradation rates of BuX and BHA decreased significantly to only 57.1% and 8.6% in the same time.

[0265] The above results indicate that both high and low temperature conditions can significantly inhibit the activity of free bacteria, and the prepared immobilized bacterial agent improves the temperature tolerance of the bacterial community and has broad development prospects in the remediation of actual contaminated sites.

[0266] Example 12 Degradation ability of immobilized bacterial agents under heavy metal stress

[0267] 1. Experimental Methods

[0268] Add Cd to 50 ml of MSM medium containing 100 mg / L BuX and BHA. 2+ , so that its concentration reaches 5 mg / L, the initial pH of the system is 7, and then 2% (volume ratio) of the free bacterial group (FB) prepared in Example 5, 2g of the immobilized bacterial agent (IB) prepared in Example 8, and 2g of the uninoculated immobilized beads (SB) prepared in Example 8 are respectively inoculated therein, and the reaction is carried out at 35°C and 150rpm for 48h, with three replicates in each group. The blank control group (CK) is not subjected to other inoculation treatments, and samples are taken at regular intervals to determine the remaining BuX and BHA contents in the solution.

[0269] 2. Experimental Results

[0270] Depend on Fig.23 It can be seen that Cd 2+ Under stress, the degradation effect of immobilized strains on pollutants was significantly better than that of free bacteria. 2+ When the concentration reached 5 mg / L, the degradation rate of BuX by IB reached more than 99% in 9 hours, while the degradation rate of FB group was only 37.6% in the same period. This difference was more obvious in BHA degradation. IB could completely degrade 100 mg / L BHA in the solution in 30 hours, while FB could only degrade 28.8% of BHA after 48 hours of culture.

[0271] Example 13 Recycling of immobilized bacterial agents

[0272] 1. Experimental Methods

[0273] Add BuX and BHA to 50mL of MSM medium with an initial pH of 7, and the final concentration of BuX and BHA is 100mg / L. Weigh 2g of immobilized bacterial agent and add it. Then put the packaged conical bottle into a 35℃ constant temperature shaker for 48h of shaking culture. Take samples at intervals and record the changes of BuX and BHA concentrations over time. After 48h, separate the immobilized beads from the culture medium, wash them 3 times with sterile saline, and transfer them to new culture medium. Cultivate them again under the above conditions and reuse them many times until the immobilized beads break.

[0274] 2. Experimental Results

[0275] result Fig.24 As shown in the figure, repeated use leads to loose internal structure of the immobilized beads, the number of embedded living cells continues to decrease, and the active sites of the immobilized material are occupied, which reduces its adsorption capacity for pollutants. Therefore, when the number of cycles increases, the time required for the immobilized bacteria to completely degrade pollutants will also increase. After multiple recycling, the mechanical strength of the immobilized beads gradually decreased until the immobilized beads broke after the fifth cycle. However, after five cycles, the immobilized beads can still completely degrade BuX and BHA with an initial concentration of 100 mg / L within 48 hours, indicating that the bacterial community after immobilization treatment is more stable and has good recycling ability.

[0276] Example 14 Storage stability of immobilized bacterial agent

[0277] 1. Experimental Methods

[0278] The immobilized bacterial agent prepared in Example 8 was stored in a refrigerator at 4°C until 0, 10, 30, 50, 70 and 90 days, and then inoculated into 50 mL of MSM culture medium (pH = 7) with an initial concentration of BuX and BHA of 100 mg / L at 35°C and 150 rpm. Samples were taken after reacting for 24 h and 48 h to determine the contents of BuX and BHA, respectively.

[0279] 2. Experimental Results

[0280] The degradation effect of immobilized bacteria on BuX and BHA after storage for a certain period of time is as follows Fig.25 As shown. The degradation rate of BuX and BHA by the freshly prepared immobilized bacteria can reach 100%, and after being stored at 4°C for 70 days, it can still maintain a degradation rate of more than 98%. When the storage time reaches 90 days, the degradation rate of BuX remains above 98%, and the degradation rate of BHA is reduced to 84.3%. The results show that the immobilized bacteria prepared by the adsorption-encapsulation method can maintain good microbial activity after long-term storage in a low-temperature environment.

[0281] Example 15: Remediation of composite contaminated soil using immobilized bacterial agents

[0282] 1. Preparation of simulated contaminated soil

[0283] The test soil was divided into two parts, one part was not treated (S1), and Cd(NO3)2 solution was added to the other part (S2) to make Cd 2+ The final concentration reached 5 mg / kg, and the soil was placed in a ventilated place for aging for one month. Then BuX and BHA were added slowly in batches and fully stirred to make the concentrations of BuX and BHA in both groups of soil reach 50 mg / kg. The composite contaminated soil was aged in the dark for two weeks.

[0284] 2. Remediation of compound contaminated soil

[0285] The two groups of aged contaminated soils S1 and S2 were divided into four equal parts, each containing 200g of soil and placed in a 500mL plastic box. To each group of soil, 5% (w / w) of the immobilized bacterial agent (IB) prepared in Example 8, 5% (w / w) of the immobilized beads (SB) not inoculated with bacteria prepared in Example 8, and 5% (V / V) of the free bacterial community (FB) prepared in Example 5 were added respectively. In the last part, 5% sterile water was added as a blank control (CK). Three parallels were set for each treatment group. During the remediation of the composite contaminated soil, water was added regularly to keep its moisture content at about 20%. Samples were taken at 0, 3, 5, 7, and 14 days after remediation. The specific grouping is shown in Table 6 below.

[0286] Table 6 Grouping of compound contaminated soil remediation

[0287]

[0288] Example 15 Effect of immobilized bacterial agent on the removal of BuX in composite contaminated soil

[0289] 1. Experimental Methods

[0290] The BuX content of the soil of each sample of Example 14 was detected by the following method:

[0291] To prevent BuX from decomposing, freeze-dry some soil as soon as possible after sampling. Accurately weigh 10g of dried soil into a 50mL centrifuge tube, add 4mL of ammonia-methanol solution (30 / 70, v / v) thereto, and vibrate at 2500rpm for 8min to achieve the purpose of full extraction. After vibration, centrifuge at 8000rpm for 10min, transfer the supernatant to a new centrifuge tube, extract the precipitate again according to the above steps, and merge the supernatants after two centrifugations. Add 50mg C18 purifier to the combined supernatant, vortex for 1min to fully adsorb impurities, centrifuge at 8000rpm for 10min, take the supernatant through a 0.22μm filter membrane, and adjust the filtrate pH to about 10.5 using formic acid, and detect BuX according to the method of reference example 4.

[0292] 2. Experimental Results

[0293] The results are as follows Fig.26 As shown in the figure, BuX content in the CK group will gradually decrease because BuX itself is unstable and its structure is relatively simple, making it easy to be used by some indigenous microorganisms. At 14 days after restoration, the BuX content in the CK group in S1 and S2 was 25.95 and 29.18 mg / kg, respectively. The higher BuX content in S2 is due to the fact that Cd 2+ It will chelate with BuX, inhibiting the natural degradation of BuX, and at the same time aggravate the toxic effect on indigenous microorganisms, interfering with their utilization of BuX.

[0294] At 14 days after restoration, the BuX contents in the FB groups of S1 and S2 were 10.82 and 20.54 mg / kg, respectively. This shows that the addition of free bacteria did promote the degradation of BuX in the soil, but the addition of Cd 2+ The presence of will significantly inhibit the degradation of BuX by free bacteria. Although the SB group was not stressed by the combined pollution, the adsorption of BuX reached saturation after 14 days of repair, and the BuX removal rate reached about 60%.

[0295] Compared with the above groups, the IB group with immobilized bacteria can effectively remove BuX in soil in both S1 and S2, and the removal rate of BuX in the IB group reached more than 90% after 14 days. This result shows that the immobilized bacteria prepared by the adsorption-encapsulation method significantly alleviates the toxic effects of pollutants on the microbial community, enhances the tolerance of the microbial community to the environment, and accelerates the removal rate of pollutants by the microbial community.

[0296] Example 15 Effect of immobilized bacterial agent on pH of composite contaminated soil

[0297] 1. Experimental Methods

[0298] The soil pH of each sample of Example 14 was detected by the following method: 2 g of air-dried soil was placed in a 50 mL centrifuge tube, and 5 mL of deionized water without CO2 was added thereto at a ratio of 1:2.5 (w / v). After sealing, the tube was placed on an oscillator at 180 rpm for 30 min, and after standing for 45 min, the pH value of the supernatant was measured with a pH meter. The average value of the three measurements was taken as the soil pH.

[0299] 2. Experimental Results

[0300] Fig. 27 It can be seen that the initial soil pH was weakly acidic, and the pH of S1 after FB restoration to 14 days was higher than that of CK group. 2+ The presence of pollutants, while the decomposition of pollutants themselves is limited, the removal of pollutants by the flora is also inhibited. At this time, there is no significant difference in pH between FB and CK. The soil pH of the IB group and the SB group continued to rise throughout the restoration process, which also shows that the alkalinity of biochar can neutralize weakly acidic soil to a certain extent, thereby increasing its pH value.

[0301] Example 16 Effect of immobilized bacterial agents on enzyme activity in composite polluted soil

[0302] 1. Pretreatment of soil samples

[0303] After sampling of each sample in Detection Example 14, part of the sample was air-dried and passed through a 200-mesh sieve to wait for the determination of its enzyme activity.

[0304] 2. Effects of immobilized bacteria on catalase activity in composite-contaminated soils

[0305] 1. Experimental methods

[0306] (1) Construction of a standard curve for catalase activity: 0, 1, 2, 3, 4, and 5 mL of 0.3% (v / v) H2O2 solution were taken in a 50 mL volumetric flask, 5 mL of sulfuric acid (1.5 mol / L) was added, the volume was made up to the mark with deionized water, and 1 mL of saturated potassium aluminum sulfate solution was added. After mixing, the absorbance of the solution was measured at 240 nm.

[0307] (2) Take 2 g of the pretreated soil sample and place it in a 50 mL centrifuge tube. Accurately pipette 40 mL of deionized water and 5 mL of 0.3% H2O2 solution into the sample. Oscillate the sample at 200 rpm on an oscillator for 20 min. Immediately add 1 mL of saturated potassium aluminum alum solution, mix well, and filter the mixture into a conical flask containing 5 mL of sulfuric acid (1.5 mol / L).

[0308] (3) Measure the OD240nm of the filtrate using UV-Vis.

[0309] This experiment requires the setting of soil-free and substrate-free controls.

[0310] 2. Experimental results

[0311] The results are as follows Fig.28 As shown in the figure, the catalase activity in the soil in the S1 group generally showed a trend of first decreasing and then increasing during the entire restoration process. On the 3rd day, the enzyme activity decreased, indicating that BuX and BHA inhibited the catalase activity in the soil. The two degrading bacteria in the immobilized bacteria need time to adapt to the new environment, after which they can use BuX and BHA in the soil as carbon sources to promote their own growth and reproduction while reducing the content of pollutants in the environment. Therefore, after 3 days, the catalase activity of the FB group and the IB group continued to increase. When the restoration lasted for 14 days, the catalase activity reached 2.14 and 2.47 mg / g, respectively. Among them, the enzyme activity of the IB group changed most significantly. Compared with CK, the catalase activity of the soil in the IB group increased by 33.36%. The increase in catalase activity in the SB group may be due to the adsorption of pollutants by the immobilized material, which weakened the effect of pollutants on the activity of indigenous microorganisms and increased the catalase activity in the soil.

[0312] 2. Effects of immobilized bacteria on urease activity in composite-contaminated soil

[0313] 1. Experimental methods

[0314] (1) Construction of urease activity standard curve: Accurately pipette 0, 1, 3, 5, 7, 9, 11, and 13 mL of 0.1 mg / mL ammonia standard solution into a 50 mL volumetric flask, add water to 20 mL. Then add 4 mL of sodium phenolate solution and 1.5 mL of sodium hypochlorite solution, mix well and let stand for 20 min. After color development, dilute to the scale line and measure its absorbance at 578 nm within 60 min.

[0315] (2) Weigh 1g of the pre-treated soil sample into a 50mL conical flask, add 0.2mL of toluene to remove the interference of microorganisms on the results. After standing for 15 minutes, add 4mL of citrate buffer solution (pH 6.7) and 2mL of 10% (W / W) urea solution to the conical flask, mix well and place in a constant temperature oscillator at 37°C and 150rpm for 24 hours. After incubation, filter it, draw 0.2mL of the filtrate into a 10mL colorimetric tube, then add 0.8mL of sodium phenol solution and 0.6mL of sodium hypochlorite solution in turn, and dilute to 10mL with water after 20 minutes of color development.

[0316] (3) Measure the absorbance of the solution at a wavelength of 578 nm within 60 min.

[0317] At the same time, soil-free and matrix-free controls were set up to eliminate the influence of the original ammonia in the soil samples on the results.

[0318] 2. Experimental results

[0319] The results are as follows Fig.29 As shown in the figure, at 14 days after restoration, the urease activities of CK, SB, FB and IB in the S1 group were 0.19, 0.40, 0.42 and 0.41 mg / (g·h), respectively, which can increase the soil urease activity. 2+ stress, the urease activity of the FB group was only 0.20 mg / (g·h), while the urease activity of the IB group reached 0.33 mg / (g·h). The results showed that the addition of immobilized bacteria can help increase soil urease activity, catalyze urea hydrolysis, promote soil nitrogen cycle, and improve soil fertility.

[0320] Example 17 Effect of immobilized bacterial agents on bacterial community structure in complex contaminated soil

[0321] 1. Experimental Methods

[0322] 24 soil samples treated to the 14th day in Example 14 were collected and sent to Guangzhou Jidi Ao Biotechnology Co., Ltd. for 16S rRNA amplicon sequencing. The V3-V4 region of the bacterial 16S rRNA gene was PCR amplified using specific primers (341F: 5'-CCTACGGGNGGCWGCAG-3'; 806R: 5'-GGACTACHVGGGTATCTAAT-3'). After extraction and purification, the amplified product was sequenced using the Illumina platform of Guangzhou Jidi Ao Biotechnology Co., Ltd.

[0323] 2. Experimental Results

[0324] 1. Changes in soil bacterial community diversity

[0325] Based on the 16S rRNA sequencing results, the Alpha diversity of the soil bacterial community in each treatment group at 14 days after restoration was performed, and the results are shown in Table 7.

[0326] Table 7 Alpha diversity index of bacteria:

[0327]

[0328] Note: The data in the table are the mean ± standard deviation of 3 parallel samples. The same letter at the end of the data in the same column indicates that there is no significant difference (P>0.05).

[0329] Shannon Diversity Index: an indicator that comprehensively measures species richness and individual distribution uniformity, often used to assess the diversity of ecosystems; Simpson Diversity Index: a diversity index that focuses on reflecting the relative richness of species, often used to describe the differences in the number of individuals between species; Chao1: a method for estimating the number of unobserved species based on sample data, suitable for situations where the sample size is small or the species distribution is uneven; Abundance-based Coverage Estimator (ACE): a method for estimating the total number of species in the entire community based on the distribution of the number of individuals of species in the sample; Coverage: refers to the proportion of species observed in the sample to the actual total number of species, used to evaluate the representativeness of the sample and the completeness of the survey.

[0330] The Coverage index represents the sequencing depth, and its values ​​are all greater than 0.98, indicating that the sequencing results can reflect the actual situation of the microorganisms in the samples.

[0331] The Chao1 and ACE indexes of the CK group in S2 were higher than those of the CK group in S1, while the Shannon and Simpson indexes were higher in the CK group in S1. 2+ The presence of heavy metals exacerbates biological toxicity, strains that cannot tolerate heavy metal stress cannot survive, the diversity of indigenous microorganisms decreases, and the abundance of dominant strains increases.

[0332] In S1, the diversity of SB and FB groups decreased compared with that of CK group, but their Chao1 and ACE indexes increased, indicating that the addition of free bacteria and immobilized bacterial agents can increase the abundance of bacterial communities.

[0333] The various indexes of the FB group in S2 increased, indicating that the free bacterial community can tolerate a certain concentration of heavy metals. The addition of free bacterial community can stimulate the activity of indigenous microorganisms, increasing their diversity and abundance.

[0334] The various indexes of the treatment group inoculated with immobilized bacteria were the highest in S1 compared with other groups, indicating that the materials used to prepare the immobilized bacteria provided a more stable growth environment for microorganisms, and the addition of foreign flora could stimulate the growth of indigenous bacteria. The immobilized bacteria prepared by combining the two helped to increase the diversity and abundance of soil bacterial communities.

[0335] 2. Beta diversity analysis

[0336] Beta diversity analysis is to compare the bacterial community composition in different treatment groups and use the distance between treatment groups to describe the similarity of species composition structure. The closer the distance, the more similar the composition structure. Fig.30 The PcoA diagram is a Bray-Curtis distance calculated based on the OUT of each treatment group. It shows that in S1, the SB and FB groups are close to each other, but far away from other groups, indicating that these two treatments can change the community structure of indigenous microorganisms, but the bacterial community structure in the soil after the two treatments is not much different. In S2, CK, SB, and FB are all close to each other, indicating that Cd 2+ Under stress, adding free bacteria or immobilized bacteria to the composite contaminated soil had little effect on its original bacterial community structure. The IB group in S1 and S2 maintained a long distance from other groups, indicating that the addition of immobilized bacteria caused a significant change in the soil bacterial community structure.

[0337] 3. Changes in abundance of soil bacterial communities

[0338] (1) Door level

[0339] The results are as follows Fig.31 As shown. At the phylum level, the bacterial composition in each treatment group in S1 and S2 was basically the same, and the dominant phyla included Proteobacteria (20.6-42.49%), Bacteroidota (14.23-32.42%), Actinobacteriota (7.93-16.73%), Chloroflexi (8.95-13.36%), Acidobacteriota (5.89-9.66%), and Firmicutes (3.26-10.66%). The proportions of each dominant phylum in different treatment groups were slightly different, among which Proteobacteria and Bacteroidota were the two main dominant phyla.

[0340] Bacteroidota accounted for the highest proportion in the CK and FB groups, accounting for 25.42% and 32.42% in the CK group of S1 and S2, and 27.68% and 26.98% in the FB. In the IB group of S1 and S2, the proportion of this phylum decreased to 14.23% and 17.81%, respectively. In the treatment group with the addition of immobilized bacteria, Proteobacteria became the most dominant phylum, accounting for 31.82% and 42.49%, respectively. Compared with the CK group, the relative abundance of this phylum increased by 54.24% and 71.74%, respectively. Based on the above results, it is believed that Proteobacteria plays a key role in the degradation of BuX and BHA, and the addition of immobilized bacteria can significantly increase the relative abundance of this phylum in the soil.

[0341] (2) Genus level

[0342] Fig.32 The relative abundance of each genera in the soil bacterial community of different treatment groups at the genus level was shown. The dominant genera mainly included Sphingomonas (9.71-15.96%), Cupriavidus (0.15-10.01%), Ramlibacter (0.86-3.46%), Pseudomonas (0.06-9.22%), Flavisolibacter (12.5-27.84%) of the Bacteroidota phylum, Bacillus (2.04-9.47%) of the Firmicutes phylum, Micromonospora (1.87-6.11%) and Streptomyces (0.69-1.78%) of the Actinobacteriota phylum.

[0343] like Fig.33 As shown in the figure, the proportion of Pseudomonas in the FB group of S1 and S2 was only 0.18% and 0.17%, respectively. In comparison, the relative abundance of Pseudomonas in the IB group increased significantly, accounting for 2.07% and 9.22% in S1 and S2, respectively. This result shows that the immobilized bacterial agent can effectively prevent the embedded microorganisms from being lost to the external environment by embedding them in a certain area of ​​space, and significantly increase their abundance. The fierce competition among the bacterial communities in S1 led to a low relative abundance of Pseudomonas, while in S2, due to Cd 2+The toxic effect of Cd caused the abundance of strains that could not tolerate heavy metal stress to decrease, and Pseudomonas became the dominant genus due to its high tolerance to Cd, and its relative abundance was further increased.

[0344] A cluster analysis was performed on the top 9 bacterial genera in relative abundance. In addition to the increase in the relative abundance of the bacterial genera contained in the immobilized bacterial agent, the addition of the immobilized bacterial agent can also stimulate the growth of certain indigenous bacteria and increase the relative abundance of these bacterial genera in the soil bacterial community.

[0345] Compared with CK, the abundance of Cupriavidus, Micromonospora, Streptomyces, and Sphingomonas in the IB group increased, among which the abundance of Cupriavidus changed most significantly. Studies have shown that strains of Cupriavidus have a good application effect in the degradation of pollutants containing N-heterocyclic structures, so it is speculated that the increase in the abundance of this genus may be due to the degradation and utilization of BHA in the soil.

[0346] Micromonospora and Streptomyces belong to the phylum Actinobacteriota. As one of the plant growth-promoting rhizobacteria (PGPR), actinomycetes have been widely confirmed to promote plant growth and improve soil fertility.

[0347] Most Sphingomonas bacteria have excellent degradation capabilities for aromatic compounds. In addition, relevant studies have shown that this genus is also a PGPR with great application potential, which promotes plant growth by regulating plant physiological metabolism and recruiting beneficial rhizosphere bacteria.

[0348] In summary, immobilized bacterial agents can weaken the toxic effects of pollutants on indigenous microorganisms, provide a suitable growth environment for beneficial bacteria, and make them the dominant bacterial genus in the soil. The enrichment of beneficial bacteria can improve the soil environmental quality to a certain extent.

Claims

1. A composition, characterized in that Contains Pseudomonas sp. and Cupriavidus sp., The Pseudomonas was deposited in Guangdong Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO.64924; The copper-greedy bacteria was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, and its preservation number is: GDMCCNo: 65117.

2. A bacterial agent, characterized in that The composition according to claim 1 is used to prepare the present invention.

3. A method for preparing an immobilized bacterial agent, characterized in that: The following steps are involved: S1. After the biochar is mixed with the composition according to claim 1, the mixture is fully adsorbed to obtain product 1; S2. The sodium alginate aqueous solution is thoroughly mixed with the product 1 to obtain the product 2; S3. Product 2 is added dropwise into CaCl2 solution to be fully solidified and obtained after purification.

4. The preparation method according to claim 3, characterized in that: In product 1, the concentration of biochar is 0.005-0.045 g / mL.

5. The preparation method according to claim 3, characterized in that: In product 2, the concentration of sodium alginate is 0.02-0.08 g / mL.

6. The preparation method according to claim 3, characterized in that: The concentration of CaCl2 solution is 0.01~0.04g / mL.

7. The preparation method according to claim 2, characterized in that: The biochar preparation method comprises the following steps: preparing corn stalk powder and calcining the powder at a high temperature of 450 to 550° C. for 3 to 5 hours.

8. The immobilized bacterial agent prepared by the preparation method according to any one of claims 3 to 7.

9. Use of the composition of claim 1 in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or in simultaneously degrading butyl xanthate and benzohydroxamic acid.

10. Use of the bacterial agent according to claim 2 or the immobilized bacterial agent according to claim 8, characterized in that: The application is one or more of the following: Preparation of products for simultaneous degradation of butyl xanthate and benzohydroxamic acid; Application in simultaneous degradation of butyl xanthate and benzohydroxamic acid; Application in remediation of soil contaminated by butyl xanthate and benzohydroxamic acid; Application of butyl xanthate and benzohydroxamic acid in remediation of polluted water bodies.

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